ES2233929T3 - GENE OF PHOSPHOLIPASE D FROM A PLANT. - Google Patents

Abstract

A cloned DNA that codes for a plant-derived phospholipase D, and a cloned DNA that controls the expression of a plant-derived phospholipase D gene.

Description

Phospholipase D gene from a plant.

Technical field

The present invention relates to a gene of the phospholipase D from a plant.

Prior art

Phospholipase D (hereinafter also referred to as "PLD") is one of the enzymes of decomposition of phospholipids that catalyzes, for example, the reaction of decomposition of lecithin to release phosphatidic acid and hill. This enzyme is known to be found in plants, animals and microorganisms. PLD is used as a reagent to measure phospholipids in blood as well as for the hydrolysis of phospholipids and for the production of derivatives by exchange reaction basic using the reversible reaction by PLD.

Purification and purification have been described partial of PLDs from plants. That is, the rice PLD purification (Men Hui Lee, 1989, Phospholipase D of rice bran, I, purification and characterization, Plant Science 59, 25-33); partial purification of rice PLD (Katsumi TAKANO, Ikuzo KAMOI and Tetsujiro OBARA, 1987, About separation and purification, and properties of rice bran phospholipase D, J. Jpn. Soc. Food Sci. Technol., 34, 8-13 (1987)); PLD purification of peanuts (Michael Heller, Nava Mozes, Irena Peri and Eddie Maes, 1974, Phospholipase D from peanut seeds, IV, Final purification and some properties of the enzyme, Biochem. Biophys., Minutes 369, 397-410); and purification of pumpkin PLD (Romy Lambrecht and Renate Ulbrich-Hofmann, 1992, A facile purification procedure of phospholipase D from cabbage and its characterization, Biol. Chem. Hoppe-seyler 373 (2), 81-88). However, nothing has been described amino acid sequences of plant PLDs and have not published the genetic analyzes of these.

On the one hand, PLD gene analyzes of microorganisms and animals have been published (patent application pending (Kokai) No. 3-187382; Adrian LM Hodgson, Phillip Bird and Ian T. Nisbet 1990, Cloning, nucleotide sequense and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis , Journal of Bacteriology 172, 1256-1261; and Japanese patent application pending (Kokai) No. 5-76357).

If a source PLD gene was available of a plant, the PLD from the plant could be produced at large scale through a genetic engineering procedure, what which would be industrially advantageous. However, as has been mentioned above, since the sequence of amino acids and the DNA sequence of plant PLD, is impossible, therefore, to genetically manipulate the PLD gene. Also, as mentioned above, even though they have been published amino acid sequences and DNA sequences from PLDs of microorganisms and animals, since they are not observed sequence homologies between the PLD genes of microorganisms or between the PLD genes of microorganisms and animals, it is difficult isolate the PLD gene from plants with the available information. In addition, since there is no information on the PLD gene in plants, an antisense DNA that suppresses the expression of the PLD gene in plants.

Description of the invention

An object of the present invention is provide a PLD gene from a plant. Other objective of the present invention is to provide an antisense DNA that can suppress the expression of the PLD gene mentioned above. Still another object of the present invention is to provide a DNA. that regulates the expression of the PLD gene from a plant.

After an intense study, those present inventors succeeded in isolating the rice PLD gene and in the sequencing of the PLD gene by purification of the PLD from rice, in determining the sequence of partial amino acids thereof, in the screening of a library of rice cDNA using the PCR product obtained as a probe using the oligonucleotides that encode the sequence of partial amino acid mentioned above, in gene cloning inserted into positive clones and gene sequencing inserted. In addition, the present inventors have succeeded in obtaining a clone of corn PLD cDNA using as probe the DNA encoding rice PLD and in the sequencing of the PLD gene of corn The present inventors have succeeded, in addition, in the isolation of a genomic DNA clone carrying the sequence regulator of the rice PLD gene and its sequencing.

That is, the present invention provides a DNA. which encodes a phospholipase D from a plant. The present invention also provides a DNA that regulates gene expression of phospholipase D from a plant.

By the present invention it was cloned, by First time, a PLD gene from a plant. Using the DNA according to the present invention, the PLD from a plant, which is useful industrially, can be produced at large Scale through a process of genetic manipulation. Further, by the present invention, a DNA was cloned for the first time which regulates the expression of the PLD gene from a plant. Thanks to this, the expression of related genes can be suppressed with lipids, thus modifying the lipids of the plant.

Best way to carry out the invention

As mentioned above, the DNA of according to the present invention encodes the PLD from a plant. In the examples included in this document, more below, the DNA encoding the rice PLD was isolated and sequenced. The deduced amino acid sequence encoded by said DNA sequencing is shown in SEQ ID NO: 2 in the List of sequences The nucleotide sequence determined so experimental that encodes the amino acid sequence shown in the SEQ ID NO: 2 is shown in SEQ ID NO: 1. Therefore, the amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence shown in SEQ ID NO: 2. In addition, in the examples described herein below were isolated a cDNA clone of the corn PLD gene using as a probe the DNA so sequenced that encodes the rice PLD and sequenced the corn PLD gene. The deduced amino acid sequence encoded by the DNA so sequenced is shown in SEQ ID NO: 4 of the Sequence Listing. The determined nucleotide sequence experimentally encoding the amino acid sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 3. Therefore, the amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 4.

In addition, in the examples described below, the Rice cDNA library was screened using DNA as a probe coding of the rice PLD thus obtained and the clones were isolated positive, followed by sequencing of the inserted DNA to get a clone that carries an inserted DNA that has the sequence of nucleotides shown in SEQ ID NO: 5. In SEQ ID NO: 5, the base 1876 is "A", which is the first "A" in the codon of start of the ATG translation of the structural gene of the PLD and the upstream direction region of the "A" (ie nucleotide 1-1875) is thought to be a regulatory region of PLD gene. The amino acid sequence encoded by the region downstream direction of nucleotide 1876 is interrupted by a intron

The DNA according to the present invention is you can obtain, for example, the procedure described in the examples below in which rice or corn is used as starting material. Alternatively, since the sequence nucleotide gene was described by the present invention, the PLD gene can be easily prepared by the procedure of the PCR using as primers a pair of oligonucleotides, each one of which hybridizes with the respective end of the DNA of according to the present invention and using the genomic DNA of Rice or corn as a template.

A PLD producing plant can be obtained inserting the DNA encoding the PLD from a plant according to the present invention within a plant vector by a conventional procedure and transforming a plant with the recombinant vector obtained by a procedure conventional.

Inserting the DNA in accordance with this invention within a vector for reverse direction plants, is You can obtain an antisense DNA from the PLD gene. Transforming a plant with said recombinant vector, the mRNA that hybridizes is produced with the mRNA formed by transcription of the intrinsic PLD gene to the plant, in such a way that gene expression can be suppressed of the intrinsic PLD for the plant. If this technique is applied to a rice plant, you get a rice plant in which you can suppress the expression of the PLD gene, so that the taste of rice

The DNA that regulates the PLD gene from A plant according to the present invention can be used in combination with the antisense DNA of the PLD gene. Through this, antisense DNA can be expressed in the same place where it expresses the gene intrinsic to the plant.

It is well known in the art that there are cases where the activity of an enzyme is retained even if the amino acid sequence of an enzyme is modified in a small region, that is, even if one or more of the amino acids in the amino acid sequence are substituted or deleted, or even if one or more amino acids are added to the amino acid sequence. The DNA encoding proteins that have such modifications and that have PLD activity are included within the scope of the present invention That is, they are also included in the scope of the present invention cloned DNA encoding sequences amino acids that have the same amino acid sequence as SEQ ID No.: 2 or SEQ ID NO: 4, except that one or more of the amino acids are substituted, deleted or added, which give the activity enzymatic of the PLD. Similarly, they are also included within the scope of the present invention the DNAs having the same nucleotide sequence as SEQ ID NO: 5 except that one or more nucleotides are substituted, deleted or added, which regulate the expression of the DNA encoding the amino acid sequence which gives the enzymatic activity of the PLD.

The modification of the DNA that causes the addition, deletion or substitution of the encoded amino acid sequence by this it can be related to site specific mutagenesis which is well known in the art (eg Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982). At the moment description, "one or more amino acids" means the number of amino acids that can be added, deleted or substituted by site-directed mutagenesis.

Site-specific mutagenesis can be carry out, for example, using an oligonucleotide primer synthetic complementary to a single stranded phage DNA except that the mutation is as follows. That is, using the synthetic oligonucleotide mentioned above as primer, is produced a complementary chain by a phage and cells bacterial hosts transform with double stranded DNA obtained. The transformed bacterial cell culture is placed in agar plates and the plates are formed from a cell simple that contains the phage. Theoretically, 50% of the new colonies contain the phage that has a simple carrier chain of the mutation and 50% of the remaining colonies contain the phage It has the original sequence. The plates obtained are subjected to then to hybridization with a synthetic probe treated with kinase at a temperature at which the probe hybridizes with the DNA which has exactly the same sequence as the DNA that has the desired mutation but not with the original DNA sequence that is not Completely complementary with the probe. Then the plates in which hybridization was observed are collected, grown and DNA is collected.

In addition to site-specific mutagenesis mentioned above, the procedures for replacement, deletion or addition of one or more amino acids without loss of Enzymatic activity includes a procedure in which the gene is deals with a mutagen and a procedure in which the gene is cut selectively, a nucleotide is removed, added or replaced selected and then the gene is ligated.

Examples

The present invention will now be described more in detail through the examples. However, this invention is not restricted to the examples described further down.

1. Purification of the PLD of bran rice

For purification, a reference was made that relates to the purification of rice bran PLD (Takano et al ., J. Jpn. Soc. Food Sci. Technol., 34, 8-13 (1987)). Enzyme activity was measured using phosphatidylcholine as a substrate and quantifying the choline generated by the choline oxidase procedure (Imamura et al ., J. Biochem. 83, 677-680 (1978)). The enzymatic reaction, however, was stopped by heat treatment at 95 ° C for 5 minutes.

That is, one liter of hexane was added to 100 g of Oryza sativa rice bran "Koshihikari" and the mixture was stirred overnight to defat the rice bran. Next, 10 g of POLYCRAL AT (brand: polyvinylpyrrolidone commercially available from GAF Chemical) and 500 mL of 10 mM Tris-HCl buffer (pH 7) containing 1 mM CaCl 2 and 5 mM 2-mercaptoethanol were added and the The resulting mixture was stirred for one hour to extract the enzyme. The extract was filtered through a strand 8 mesh and the filtrate was centrifuged at 15,000 xg for 20 minutes. The intermediate layer was collected as a crude extract. The crude extract was treated with ammonium sulfate (65% saturation) and the precipitates formed were collected by centrifugation (15,000 xg, 20 minutes). The precipitates were dissolved and dialyzed against the buffer described above. After dialysis, the precipitates were removed by centrifugation to obtain a fraction of ammonium sulfate.

The ammonium sulfate fraction was applied in a DEAE-cellulose column (commercially available of Whattman) (2.0 x 10 cm) balanced with a buffer A (10 mM Tris-HCl pH 7, 1 mM CaCl2, 1 mM 2-mercaptoethanol). After washing the spine with approximately 100 mL of buffer A containing 50 mM NaCl, it was carried out the elution with 120 mL of buffer A which had a Linear NaCl gradient of approximately 50 to 350 mM. The PLD is eluted in the vicinity of 0.2 M NaCl. The fractions they exhibited PLD activity were collected as an eluted solution (DEAE-cellulose).

To the eluted solution (DEAE-cellulose), 3M ammonium sulfate was added to a concentration of 1 M and the resulting was applied to a column of Phenyl Sepharose (commercially available from PHARMACIA, 2.6x10 cm). Next, the column was eluted with 240 mL of buffer A which had an ammonium sulfate gradient of 1.0 to 0. It eluted the PLD in the vicinity of 0.1 M ammonium sulfate. fractions that exhibited PLD activity were collected and dialyzed against buffer A to obtain an eluted solution (Phenyl Sepharosa).

The eluted solution (Phenyl Sepharose) was applied to a MonoQ column (anion exchange column, available commercially from PHARMACIA, 16 x 10 cm) and then carried carried out the elution with 150 mL of buffer A which had a gradient of 50-350 mM NaCl. PLD eluted at concentrations of 210 mM NaCl at 235 mM. The fractions that exhibited activity PLD were collected and dialyzed against buffer A to obtain a eluted solution (Mono Q 1st).

The eluted solution (Mono Q 1st) was concentrated to 0.5 mL by centrifugal ultrafiltration and the resulting was applied to a Superose 6 column (commercially available from PHARMACIA, 1.0 x 30 cm) equilibrated with buffer A containing 0.1 M NaCl and the column was eluted with the same buffer. Molecular weight of the PLD was estimated at 78 kDa. The fractions they exhibited PLD activity were collected as an eluted solution (Superose 6).

To the eluted solution (Superose 6), were added 2.5 mL of CARRIER AMPHOLITE (commercially available from PHARMACIA ph 4.0-6.0) and distilled water to a final volume 50 mL The resulting was then subjected to focus isoelectric using Rotofore (commercially available from BIORAD). The electrophoresis was carried out at 2 ° C under a power 12 W constant for 4 hours. PLD activity was observed in the pH proximity 4.9. Fractions exhibiting were collected PLD activity and the solution obtained was dialyzed against buffer A to obtain an isoelectric focusing fraction.

The isoelectric focusing fraction was applied to a Mono Q column (commercially available from PHARMACIA, 0.5 x 5 cm) and elution was carried out using a buffer A that had a Linear NaCl gradient from 50 mM to 350 mM. The PLD was eluted in the proximity of NaCl 210 mM and 235 mM. The two fractions they exhibited PLD activity were collected as eluted solutions (Mono Q 2nd-II).

The purities of the eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II) se determined by gel electrophoresis SDS-polyacrylamide (Laemmli (1970)). After the electrophoresis, the gels were stained with COOMASSIE BRILLIANT BLUE R250 With any of the solutions eluted, a main band at the molecular weight position of 82 kDa. Be observed a single band with the eluted solution (Mono Q 2nd-II).

Through the purification described above, the eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II) were purified 380 times and 760 times respectively, with respect to the crude extract.

The two fractions were analyzed by properties of the enzymes contained in them. The results are shown in Table 1. Buffer solutions used to measure the optimal pH were sodium acetate (pH 4-6), MES-NaOH (pH 5.5-7.0) and Tris-HCl (pH 7-9), all having a concentration of 100 mM. PH stability means the range in which the decrease in activity was not observed after leaving The enzyme will be at 25 ° C for 30 minutes. Thermal stability is measured by measuring the remaining activity after leaving the enzyme be at 4 ° C, 25 ° C, 37 ° C or 50 ° C for 30 minutes. Specificity of substrate was measured at a substrate concentration of 5 mM and was expressed as the relative activity taking the enzymatic activity with respect to phosphatidylcholine as 100.

TABLE 1

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one

2. Proof that the purified protein is PLD

In the same way as in determining the purity, eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II) were subjected, separately, to SDS-polyacrylamide gel electrophoresis and each of the gels was transferred to a PVDF membrane (commercially available from MILLIPORE) followed by membrane staining. Be cut the band corresponding to the protein that had a weight 82 kDa molecular was cut and sequence determined amino acid of the N-terminal region of the protein. The amino acid sequence could be determined for both proteins until the tenth amino acid residue and both proteins had the same sequence as follows:

Val Gly Lys Gly Ala Thr Lys Val Tyr Be

Although the relationship was unknown between the 82 kDa proteins that existed in the two fractions that they had enzymatic activity, it was thought that at least the homology of the amino acid sequences thereof was high. In this way, it was thought that there were no problems even if one mixture of these factors was used as an antigen for the production of an antibody.

A mixture of the eluted solutions was subjected (Mono Q 2nd-I and Mono Q 2nd-II) a SDS-polyacrylamide gel electrophoresis using 7.5% acrylamide and the gel was stained with COOMASSIE BRILLIANT BLUE R250 The band containing the 82 kDa protein was cut and the proteins were recovered by electroelution (25 mM Tris, 192 mM glycine, 0.025% SDS, 100 V, 10 hours). After extracting SDS by electrodialysis (15 mM ammonium bicarbonate, 200 V, 5 hours), the resulting was cold dried. Electroelution and electrophoresis were carried out using BIOTRAP (available commercially from SCHLEICHER & SCHUELL).

Rabbits were immunized with the 82 protein Highly purified kDa by the procedure described further above. Immunization was carried out by administering 50 µg of protein per time in 7 day intervals. Using serum blood before immunization and after the third immunization, immunotitration was carried out. This is the PLD solution containing 8.6 x 10-3 enzyme units, was mixed with 0 to 50 µL of serum before immunization or after the third immunization, 50 µL of 250 mM Tris-HCl (pH 7.0), 5 µL of CaCl 2 50 mM, 50 µL of 0.2% TRITON X-100 (brand) and water balance to a final volume of 250 µL and the mixture is Let it stand at room temperature for 2.5 hours. To the mix resulting, 200 µL of Protein A SEPHAROSE was added (commercially available from PHARMACIA) and the resulting was stirred vigorously at room temperature for 2 hours. The resulting centrifuged (500 x g, 5 minutes) and activity was measured Enzymatic of the supernatant. Taking enzymatic activity when serum was not added as 100%, the enzymatic activities measured at add 20 µL or 50 µL of serum before immunization, were 95% and 88%, respectively, while the activities enzymatic measured by adding 20 µL or 50 µL of serum after immunization they were 75% and 30%, respectively. These results proved that the 82 kDa protein is PLD.

3. Determination of the amino acid sequence internal

The PLD protein was fragmented by fragmentation of the protein in a gel (Cleveland et al ., J. Biol. Chem., 252, 1102 (1977)). The gel containing the PLD protein, which was cut following the same procedure as described in 2, was inserted into a well of stacking gel (in English " stacking gel ") in a 15% acrylamide gel prepared for the separation of peptides . Staphylococcus aureus V8 protease (commercially available from WAKO PURE CHEMICAL INDUSTRIES, LTD) was charged in an amount of 1/10 of the PLD protein and electrophoresis was initiated. Eletrophoresis was interrupted when bromophenol blue reached the center of the packing gel and 30 minutes later electrophoresis was restarted. After electrophoresis, the gel was transferred to a PVDF membrane and the PVDF membrane was stained. The clear bands were observed in the positions corresponding to the molecular weights 20, 14, 13, 11 and 10 kDa. The bands corresponding to the molecular weights 20, 14 and 13 kDa were cut and the amino acid sequences of the peptide fragments contained in the bands were determined with a protein sequencer. The sequences are as follows:

2

(In the sequences, "?" Means the residue that could not be identified and the amino acid residue in parentheses it is one that can be another amino acid residue with a probability considerable).

4. Preparation of the seed cDNA library immature rice

Total RNAs were prepared from the immature seeds 5 days after flowering, extracting the RNAs by the SDS-phenol method and precipitating the extract with lithium chloride. RNA poly (A) + were prepared using OLIGOTEX-dT30 (available commercially from TAKARA SHUZO) according to the instructions of the maker. For cDNA cloning, the cDNA of SYNTHESIS SYSTEM PLUS (commercially available from AMERSHAM) and CDNA \ lambdagt10 CLONING SYSTEM (commercially available from AMERSHAM). As a cloning vector, the vector λZAPII (commercially available from STRATAGENE) and as host cells the XL1-Blue

5. Preparation of the probes

Oligonucleotides were synthesized corresponding to the amino acid sequence of the PLD with a DNA synthesizer (commercially available from APPLIED BIOSYSTEMS). The sequences thereof and the sequences of Corresponding amino acids are described below.

20KF: 5 'AAYTAYTTYCAYGG 3'

20KR1: 5 ' RTCRTCRTCNGGRTT 3'

(where R represents purine bases, this is A or G; Y represents pyrimidine bases, that is, T or C; and N represents G, A, T or C).

20KF is a mixture of 32 types of oligonucleotides each of which encoding the sequence of amino acids of

Asn Tyr Phe His Gly

that was found in the peptide that It had a molecular weight of 20kDa and 20KR1 is a mixture of 128 types of oligonucleotides each of which encoding the sequence of amino acids from

Asn Pro Asp Asp (Asp)

that was found in it peptide

The cDNA synthesis reaction was carried out. in a mixture of 10 ng of poly (A) + RNA, 0.3 µg of random hexamer (dN6), 10 U of Rnasa inhibitor (RNAGuard, commercially available from PHARMACIA), 1 mM each of dATP, dCTP, dGTP and dTTP, 1 x PCR buffer (commercially available from TAKARA SHUZO), 50 mm of magnesium chloride and 100 U of transcriptase Reverse (M-MuLV RTase, commercially available from BRL), the total volume of the reaction mixture being 10 µL. The reaction was carried out at 37 ° C for 30 minutes and the mixture was then heated at 95 ° C for 5 minutes, followed by the retention of the resulting mixture on ice.

The polymerase chain reaction (PCR) is carried out using the cDNA described above as template and 20KF and 20KR1 as primers. The reaction was carried out. using 10 µL of the cDNA synthesis reaction mixture, a mixture of 50 pmol of each of the primers, 200 µM of each of dATP, dCTP, dGTP and dTTP, 1 x PCR buffer (available commercially from TAKARA SHUZO) and 2.5 U of DNA polymerase AmpliTaq (commercially available from TAKARA SHUZO), the volume total of the reaction mixture being 50 µL. Was repeated 30 times a cycle of 94 ° C for 1 minute / 72 ° C for 2.5 minutes in a DNA THERMOCICLATOR (commercially available from PERKIN ELMER CETUS).

The PCR product was separated on an agarose gel. at 2%. Various fragments were detected by staining the gel with bromide of ethidium One of them had a size of 94 bp which is the expected size.

The PCR fragment was extracted from the gel and was subcloned into plasmid pUC19. The subcloned PCR fragment is sequenced using the dideoXi procedure using the kit T7 sequencing (commercially available from PHARMACIA). Between the two primers, a nucleotide sequence coding for the expected amino acid sequence. Nucleotide sequence between the two primers and the amino acid sequence encoded by This is as follows:

C TCT GAC GTC AAC TGT GTT CTA TGC CCT CGC

Be Asp Val Asn Cys Val Leu Cys Pro Arg

Isotope P32 was incorporated (available commercially from AMERSHAM) within the oligonucleotide described further above using the marking kit at the 5 'end of the DNA MEGALABEL (commercially available from TAKARA SHUZO) to obtain a radioactive oligonucleotide probe.

6. Screening of the clone containing the PLD gene

Using the radioactive oligonucleotide described above, a cDNA library was screened. The hybridization solution was 0.5 M sodium phosphate buffer (pH 7.2) containing 7% SDS, 1mM EDTA and 100 Mg / ml salmon sperm DNA and hybridization was carried out at 45 ° C for 16 hours after adding the probe to this solution. The wash solution contained 0.3 M NaCl and 30 mM sodium citrate and the washing was carried out twice at 45 ° C for 20 minutes. Positive plaques were isolated and subcloned in vivo into plasmid pBluescript (commercially available from STRATAGENE) according to the instruction of the manufacturer of the cloning vector? LambZAPII. The nucleotide sequence was determined and the coding region of the internal amino acid sequence determined at 3 existed.

7. Sequencing of the 5 'terminal Region

Since a clone containing the full length DNA could not be isolated, a DNA fragment containing the 5 'terminal region (Edwards et al ., Nucleic Acids Res., 19, 5227-5232) was prepared by the RACE method. (1991)). A 5'-AmpliFINDER RACE kit (commercially available from CLONETECH) was used according to the instructions included with the product. The oligoDNA were prepared and PCR was carried out using the mRNA prepared by the method described in 4 as a template. The PCR product was subcloned into the PCRII vector (commercially available from INVITROGEN) and sequenced by the dideoxy method. As a result, it was estimated that the translation was initiated from nucleotide 182 shown in SEQ ID NO: 1 due to an end codon that exists upstream of it of 36 bp.

8. Preparation of the cDNA clone coding for the PLD from corn

The corn cDNA clone was obtained by procedure described below using the DNA encoding the PLD of rice as a probe.

Using the suspended cultured cells achieved by culturing a callus from an immature maize embryo generated by inbreeding Mo17 (commercially available from MIKE BRAYTON SEDES, INC.) In a liquid culture medium, a cDNA library was prepared by the procedure described in 4. However, the λ vector (commercially available from AMERSHAM) was used as the cloning vector and used as NM-514 host cells (commercially available from AMERSHAM). Using the rice PLD cDNA as a probe, positive plaques were isolated by the procedure described in 6. Phage DNA was prepared according to the instructions of the cloning vector manufacturer. Phage DNA was digested with a Kpn I restriction enzyme and the resulting subcloned into a plasmid pBluescript. The nucleotide sequence was determined by the dideoxy method as described in 6.

9. Isolation of the genomic clone of the PLD corresponding to the cDNA of the PLD and identification of the region promotion girl

To isolate a genomic DNA clone carrying the regulatory sequence of the PLD gene corresponding to the 6-sequenced PLD cDNA, which was cloned into a plasmid pBluescript, a Koshihikari rice genomic library was prepared. This was accomplished by partial digestion of the Koshihikari leaf DNA with Mbo I, purification of the fractions having sizes of 16-20 kb by centrifugation with sucrose gradient and using lambda DASH II (commercially available from STRATAGENE) and GigapackII Gold (commercially available from STRATAGENE). Using the cDNA clone of the PLD as a probe, the genomic library was screened. Screening was carried out as described in 6. However, hybridization was carried out at 65 ° C for 16 hours, the wash solution was 0.5 x SSC containing 0.1% SDS and the wash was Run twice at 65 ° C for 20 minutes. The nucleotide sequence of the hybridized genomic clone was determined by the dideoxy method. As a result, there was a region homologous to the cDNA sequence determined in 6.

The transcription start site is determined by the procedure described in 7. In the vicinity to the transcription start site, the box of consensus sequence "TATA". The translation start site ATG was determined as the codon above in the reading frame of clone translation and as the ATG codon that is first accessible in the mRNA synthesized in rice.

A part of the clone DNA sequence Genomic hybridized with the cDNA clone is shown in SEQ ID NO: 5. In the genomic DNA sequence, a framework of reading that started at the translation start codon, which overlap with the corresponding cDNA sequence. The promoter region address is located upstream of the start codon of the ATG translation and start immediately above this.

SEQ ID NO: 1

SEQUENCE LENGTH: 3040

SEQUENCE TYPE: NUCLEIC ACID

TYPE OF MOLECULE: cDNA to mRNA

ORIGINAL SOURCE ORGANISM: Oryza sativa

SEQUENCE DESCRIPTION

3

4

5

6

7

8

9

SEQ ID NO: 2

SEQUENCE LENGTH: 812

SEQUENCE TYPE: AMINO ACID

TYPE OF MOLECULE: Protein

ORIGINAL SOURCE ORGANISM: Oryza sativa

SEQUENCE DESCRIPTION

10

eleven

12

13

14

SEQ ID NO: 3

SEQUENCE LENGTH: 2804

SEQUENCE TYPE: NUCLEIC ACID

TYPE OF MOLECULE: cDNA to mRNA

ORIGINAL SOURCE ORGANISM: Maíz Zea

SEQUENCE DESCRIPTION

fifteen

16

17

18

19

twenty

twenty-one

SEQ ID NO: 4

SEQUENCE LENGTH: 812

SEQUENCE TYPE: AMINO ACID

TYPE OF MOLECULE: Protein

ORIGINAL SOURCE ORGANISM: Maíz Zea

SEQUENCE DESCRIPTION

22

2. 3

24

25

26

SEQ ID NO: 5

SEQUENCE LENGTH: 2799

SEQUENCE TYPE: NUCLEIC ACID

TYPE OF MOLECULE: Genomic DNA

ORIGINAL SOURCE ORGANISM: Oryza sativa

SEQUENCE DESCRIPTION

27

28

29

Claims (1)

1.DNA, which encodes a phospholipase D enzyme, which It comprises the following sequences: 30